The objective of this study is to understand the distribution and variation characteristics of PWL gene family in different Magnaporthe oryzae strains, and to provide a basis for studying the genetic diversity and specificity of different M. oryzae strains.

A total of 8 pairs of specific primers were designed for the promoter region and CDS region of the PWL gene family by referring to the avirulence gene sequence published in NCBI. DNA was extracted from 397 single spore isolates of M. oryzae collected from different regions of Heilongjiang Province and Hainan Province in 2020. PCR amplification of avirulence genes was conducted and the genes were detected by agarose gel electrophoresis. Representative strains from different regions were selected from the test results to sequence and analyze the amplified fragments. The sequencing results were compared with the base and amino acid sequences of the corresponding avirulence gene promoter region and CDS region in NCBI.

In the results of PCR electrophoresis, PWL1 was not detected in all strains. The specific fragments of PWL2, PWL3 and PWL4 were amplified in Heilongjiang Province and Hainan Province, indicating that these three genes were distributed in both provinces and had different distribution frequencies and variation types. Among them, PWL2 had the highest distribution frequency in Heilongjiang Province and Hainan Province, which were 98.14% and 100%, respectively. The distribution frequencies of PWL3 and PWL4 in the two provinces were significantly different. The frequencies of these two genes in Heilongjiang strains were 89.30% and 82.79%, respectively, while in Hainan strains were 5.49%. Through the analysis of avirulence gene combinations, the results showed that the combination types could be divided into six types, namely PWL(f), PWL2, PWL3, PWL2+PWL3, PWL2+PWL4, PWL2+PWL3+PWL4. Among them, Heilongjiang strains contained all combination types, and Hainan strains contained only two, indicating that Heilongjiang strains were more abundant in avirulent genotypes than Hainan strains. By sequencing the PCR products of the PWL gene family, it was found that the PWL gene family had abundant variation sites in the promoter region and CDS region, and was divided into nine types with point mutations and deletions as the main variation types, and the variation types of strains from different populations were specific and consistent. Among them, five variant types PWL2- (1, 2, 3, 4, 5) were detected in PWL2, base sequence changes led to amino acid sequence missense mutations; two mutation types were detected in PWL3 and PWL4, which were PWL3- (1, 2) and PWL4- (1, 2), respectively. Both of them had frameshift mutations that caused changes in the following amino acids.

The distribution and variation types of PWL gene family in M. oryzae strains from different populations have regional differences, and the variation sites are abundant.

By detecting the distribution and variation characteristics of the AVR-Pita family in different Magnaporthe oryzae strains from Heilongjiang Province and Hainan Province, the pathogenic phenotypes of the variation types were understood, which provided a reference for the screening and breeding of disease-resistant germplasm in the region.

Six pairs of specific primers were designed for the promoter region and CDS region by referring to the AVR-Pita family sequence published in NCBI. DNA was extracted from 397 single spore strains of M. oryzae collected from different regions of Heilongjiang Province and Hainan Province in 2020, and PCR amplification was performed. By electrophoresis test results, representative strains from different regions were selected to sequence the amplified fragments. The sequencing results were compared with the corresponding base and amino acid sequences in NCBI, and the avirulence function of different variation types of M. oryzae strains was verified by using rice resistant single gene lines.

In the PCR electrophoresis results, the M. oryzae strains in Heilongjiang Province carried all the genes to be tested, which were widely distributed and had a high detection frequency. However, only AVR-Pita1 and AVR-Pita2 were carried by M. oryzae strains in Hainan Province, and concentrated in low frequency. The results of avirulence gene composition analysis showed that the strains of M. oryzae in Heilongjiang were complex and diverse, and the genotypes were more abundant than those in Hainan. The sequencing results of PCR products showed that the AVR-Pita family was divided into 19 types with point mutation, insertion and deletion as the main variation types, and the variation types of strains from different M. oryzae populations were specific. Ten variation types were detected in AVR-Pita1, among which AVR-Pita1- (1-5) was a unique variation type of M. oryzae strains in Heilongjiang Province, and AVR-Pita1- (6-10) was a unique variation type of M. oryzae strains in Hainan Province. After functional verification of these ten variation types, it was found that the avirulence functions were lost. Eight variation types were detected in AVR-Pita2, among which AVR-Pita2- (1-4) was a unique variation type of M. oryzae strains in Heilongjiang Province, and AVR-Pita2- (5-8) was a unique variation type of M. oryzae strains in Hainan Province. After functional verification, it was found that the avirulence functions were lost. Only one variation type (AVR-Pita3-1) was detected in AVR-Pita3 in Heilongjiang Province.

The AVR-Pita family in Heilongjiang and Hainan populations were mutated alleles. After pathogenicity identification, all mutation types could not be identified by the corresponding resistance genes Pi-ta and Pi-ta². Therefore, the resistance genes Pi-ta and Pi-ta² can be used to polymerize other resistance genes to ensure the disease resistance of varieties in the process of disease resistance breeding and utilization of rice blast in Heilongjiang Province and Hainan Province. At the same time, the distribution and variation types of AVR-Pita family in M. oryzae strains from different geographical sources are specific.